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Plasmid purification
Plasmid purification using Invitrogen's PureLink Quick Plasmid Prep Kit The PureLink™ Quick Plasmid Miniprep Kit is designed to isolate high quality plasmid DNA (up to 30 µg) from E. coli cells in 30-45 minutes. Cells are lysed using an alkaline/SDS procedure. The lysate is then applied to a silica membrane column that selectively binds plasmid DNA. Contaminants are removed with Wash Buffers. The plasmid DNA is eluted in TE Buffer and is suitable for all routine downstream applications. The PureLink™ Quick Plasmid Miniprep Kit can be used with a centrifuge or a vacuum manifold. Before Starting *Add RNase A to Resuspension Buffer (R3) according to instructions on the label. Mix well. Mark on the label that RNase has been added and store at 4°C. *Add 96-100% ethanol to wash buffer (W9) and wash buffer (W10) according to the instructions on each label. Mix well. Mark on the labels that the ethanol is added and store at room temperature. *If lysis buffer (L7) contains salt precipitates, warm the buffer in a 37°C water bath for a few minutes until precipitates dissolve. Do not shake the buffer. Preparing cell lysate #Pellet 2ml of an overnight culture of'' E. coli'' in LB medium by centrifugation at 10000 x g for 5 minutes. Thoroughly remove all medium from the cell pellet. #Completely resuspend the pellet in 250ul Resuspension Buffer (R3) with RNase A. No cell clumps should remain. #Add 250ul Lysis Buffer (L7) to cells. Mix gently by inverting the capped tube 5 times.Do not vortex #Incubate for 5 minutes at room temperature. Do not overincubate. #Add 350ul Precipitation buffer (N4), Mix immediately by inverting the tube until the solution is homogeneous. For lage pellets, shake more vigourously. #Centrifuge the mixture at 12000 x g for 10 minutes at room temperature to clarify the lysate from the cell debris. Binding DNA #Preheat an aliquot of TE buffer to 70°C. #Load the supernatant from step 6 above (~850ul) onto a Spin Column placed in a 2ml wash tube. #Centrifuge at 12000 x g for 1 minute. Discard the flow through and place the column back into the wash tube. #Add 500ul wash buffer (W10) with ethanol to the column. Incubate for 1 minute at room temperature. Centrifuge at 12000 x g for 1 minute. Discard the flow-through and place the column back into the wash tube. #Add 700ul Wash Buffer (W9) with ethanol to the column. #Centrifuge at 12000 x g for 1 minute. Discard the flow-through and place the column back into the wash tube. #Centrifuge at 12000 x g for 1 minute to remove any residual wash buffer (W9). Discard the wash tube with the flow-through. Eluting the DNA #Place the spin column in a clean 1.5-ml recovery tube. #Add 75ul of TE buffer (TE, preheated to 70°C) to the center of the column. #Incubate for 1 minute at room temperature. #Centrifuge at 12000 x g for 2 minutes. #The recovery tube contains the purified plasmid DNA. Discard the column. Storing DNA *Store the purified DNA at –20°C or use DNA for the desired downstream application. *To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at –20°C for long-term storage. Plasmid purification using Invitrogen's PureLink HiPure Plasmid DNA Purification Kit The PureLink HiPure Plasmid DNA Miniprep Kit allows purification of up to 30 ug of high quality plasmid DNA from 1–3 mL overnight E. coli cultures in ~1 hour when cloning high copy number plasmids. Before beginning Verify that RNase A is added to the Resuspension Buffer (R3) and that no precipitate has formed in the Lysis Buffer (L7). Required materials *Overnight culture of transformed E. coli cells *Isopropanol *70% ethanol *Sterile, microcentrifuge tubes *PureLink Nucleic Acid Purification Rack *Microcentrifuge capable of centrifuging at >12,000 × g *Resuspension Buffer (R3) with RNase A *Lysis Buffer (L7) *Precipitation Buffer (N3) *Equilibration Buffer (EQ1) *Wash Buffer (W8) *Elution Buffer (E4) *TE Buffer (TE) *PureLink HiPure Mini Columns Equilibrating the columns Place the PureLink HiPure Mini column on the PureLink Nucleic Acid Purification Rack (see the manual supplied with the rack for more details). Apply 2 mL Equilibration Buffer (EQ1) to the column. Allow the solution in the column to drain by gravity flow. Proceed to lysate preparation while the column is equilibrating. Preparing cell lysate #For high copy number plasmids, use 1–3 mL of an overnight LB culture per sample in a in a microcentrifuge tube. Note: When using 2–3 mL of culture, pellet 1–1.5 mL culture twice in the same microcentrifuge tube. If you are using >5 mL of culture volume of high copy plasmids, add twice the amount of Resuspension Buffer (R3) with RNase A, Lysis Buffer (L7), and Precipitation Buffer (N3) as directed in Steps 3, 4, and 5, below for best results. For low copy number plasmids, use 10–15 mL of an overnight LB culture per sample in a 15-mL disposable tube. #Harvest the cells by centrifuging the overnight LB culture at 4,000 × g for 5–10 minutes. Remove all medium. #Add 0.4 mL Resuspension Buffer (R3) with RNase A to the pellet and resuspend cells until homogeneous. Note: If cells were resuspended in a 15-mL disposable tube, then transfer the cells in a microcentrifuge tube. #Add 0.4 mL Lysis Buffer (L7). Mix gently by inverting the capped tube until the lysate mixture is thoroughly homogenous. Do not vortex. Incubate at room temperature for 5 minutes. Note: Do not allow lysis to proceed for more than 5 minutes. #Add 0.4 mL Precipitation Buffer (N3) and mix immediately by inverting the tube until the mixture is thoroughly homogeneous. Do not vortex. #Centrifuge the lysate at >12,000 × g for 10 minutes at room temperature. Note: If the pellet does not adhere to the bottom of the tube, incubate the tube at room temperature for 5 minutes to allow the separation of lysate and gelatinous pellet. Pipette the clear lysate into another sterile tube and centrifuge at >12,000 × g for 5 minutes at room temperature to remove any remaining cellular debris. #Proceed to Binding and Washing DNA Binding and washing DNA #Load the supernatant from Step 6 (previous page) onto the equilibrated column. Allow the solution in the column to drain by gravity flow. #Wash the column twice with 2.5 mL Wash Buffer (W8). Allow the solution in the column to drain by gravity flow after each wash. Discard the flow-through. #Proceed to Eluting and Precipitating DNA. Eluting and precipitating DNA #Place a sterile microcentrifuge tube (elution tube) under the column. #Add 0.9 mL Elution Buffer (E4) to the column to elute the DNA. Allow the solution to drain by gravity flow. Do not force out any remaining solution. The elution tube contains the purified DNA. Discard the column. #Add 0.63 mL isopropanol to the elution tube. Mix well. #Centrifuge the elution tube at >12,000 × g for 30 minutes at 4°C. Carefully remove and discard the supernatant. #Resuspend the DNA pellet in 1 mL 70% ethanol. #Centrifuge at >12,000 × g for 5 minutes at 4°C. Carefully remove and discard the supernatant. #Air-dry the pellet for 10 minutes. #Resuspend the DNA pellet in 50 μL TE Buffer (TE) Storing DNA *Store the purified DNA at –20°C or use DNA for the desired downstream application. *To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at –20°C for long-term storage. Verifying successful ligation and transformation Before using purified plasmid DNA in downstream applications, it's a good idea to ensure that your PCR fragment was successfully ligated into the vector. This can be achieved relatively easily as outlined below: DNA Digest The pGEM-T Easy Vector multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BstZI, and NotI, providing three single-enzyme digestions for release of the insert. The pGEM-T Vector cloning region is flanked by recognitions sites for the enzyme BstZI. The following protocol outlines how to use EcoRI to release the insert from the pGEM-T Easy Vector: #Add >1ug (5ul) of purified plasmid DNA to a sterile 1.5-ml microcentrifuge tube on ice. #Prepare the following master mix on ice (values are for one reaction): ##1 ul EcoRI (10U/ul) ##2 ul 10 X Buffer H ##12 ul sterile water to a final volume of 15 ul #Aliquot 15ul of the master mix into each of the tubes prepared in step 1. #Incubate at 37°C for 1 hour. #Inactivate by incubating at 70°C for 10 minutes. #Add 10ul of the digestion mixture to 2ul of 6 X loading buffer. Load into an agarose gel and analyze by electrophoresis. If ligation was succesful, there should be two bands in each lane. One band is the vector (~3kb) and the other band is the insert, and its size should correspond to the size of the PCR fragment you began with. Category:Lab techniques